Clathrin coated vesicles (CCVs) carry out receptor-mediated endocytosis (RME) at the plasma membrane, where they drive the uptake of receptor bound nutrients, hormones and proteins targeted for degradation. Clathrin is shaped like a "three-legged" pinwheel to fit its function of forming honeycomb lattices around transport vesicles. The long-term goal of this proposal is to define and validate productive atomic contacts between lattice-bound clathrin molecules to elucidate the fundamental principles of how clathrin function is regulated in cells. The first specific aim is to determine the crystal structure of the clathrin trimerization domain, where the three filamentous legs meet, to complete the atomic level reconstruction of the entire clathrin molecule. Trimerization domain crystals are highly ordered, diffracting between 1.7-2.3 A at Argonne and at the Advanced Light Source. Selenomethionine crystals have been prepared to solve the phasing problem by MAD analysis, while heavy metal replacement and "cryo-halide" soaking methods are being performed as alternative phasing strategies. The X-ray model of trimerization domain will then be docked into the 21 A cryo-EM image of the clathrin barrel (with Barbara Pearse, Corrine Smith and Alan Roseman) to identify productive molecular contacts between the trimerization domain and segments of other clathrin molecules in the lattice. Revealed interactions will be tested biochemically and validated in living cells. The second aim is to explore how cysteines in the trimerization domain stabilize this structure by using structural information from Aim 1 to design mutants to test specific linkages. The third aim explores whether there is a relationship between the cysteines in the trimerization domain and those in light chain subunit to understand why these two groups of cysteines impact regulation of clathrin assembly. The experimental approach is to determine whether light chains can confer structural stability to trimerization domain constructs weakened by mutations made in Aim 2. The final aim of this proposal is to test the possibility that light chain control of lattice formation might involve the transfer of the light chain EED Ph switch between an "active" and "inactive" position on the proximal domain of clathrin. The approach is to use light chain fragments and molecule mimics to ferret out these putative sites and to use X-ray crystallography to try to capture the orientation of light chain fragments bound to the proximal region. Understanding the structural basis for clathrin function is central to defining the role of RME malfunction in hypercholesterolemia and heart disease, as well as understanding the mechanism of virus infection.